Project Summary: End-stage lung diseases are a major cause of morbidity and mortality worldwide. Lung transplantation is an excellent treatment option for patients with this condition, yet 50% of recipients die within five years due to the development of obliterative bronchiolitis (OB) in the allograft. Epithelial stem cell depletion is suggested to contribute to the development of OB; however, there is little research being performed to test this hypothesis, primarily due to the lack of animal models for OB that develop allograft pathology resembling that seen in humans. We recently developed a novel orthotopic lung transplant model in the ferret that models human OB very well. Using this model, we have shown for the first time in ferret and human allografts that the number of clonogenic K5+p63+ basal stem cells (BSCs) progressively declines in proximal and distal airways of the allograft as the severity of OB increases. Additionally, our research is the first to demonstrate that the proximal airway submucosal gland (SMG), a facultative niche for BSCs in the surface airway epithelium (SAE), is an early target of immune destruction in human and ferret allograft airways. In mice, the SMG stem cell niche serves only the trachea; however, in larger mammals such as humans and ferrets, SMGs are present throughout the cartilaginous airways. Using lineage tracing, we have shown that the myoepithelial cells (MECs) of SMGs are precursors of multipotent K5+p63+ BSCs in the SAE. During the development of OB, the destruction of SMG stem cell niches in the allograft occurs simultaneously with phenotypic and functional changes to multipotent K5+p63+ BSCs in the SAE. We hypothesize that destruction of the denervated SMG in the allograft, and thus depletion of MECs, leads to a decline in multipotent K5+p63+ BSCs in the SAE, and to increases in committed multipotent (K5+p63+K14+), bipotent (K5+K14+, p63+K14+) and unipotent (K14+) basal cells, all of which have a reduced capacity for self-renewal. The objective of the proposed research is to determine the functional significance of the phenotypic changes in the MECS and the lineage-committed basal cells and how denervation of the SMG alters Wnt signaling (Lef-1/TCF1) required for both the maintenance of the SMG stem cell niche and lineage commitment of glandular MECs to SAE BSCs in the setting of injury. Additionally, a major preclinical objective of this proposal is to elucidate the ability of stem cells to engraft into a transplanted lung and repair injury, thus laying the foundation for the development of stem cell therapy to delay or prevent OB in lung allografts. We will achieve these objectives by addressing the following specific aims: 1) Determine how destruction of the SMG stem cell niche contributes to depletion of K5+p63+ BSCs. 2) Identify consequences of lung denervation on airway stem cells and their niches. 3) Determine the potential for airway stem cell transplantation in preventing or delaying OB. We expect that delineating the mechanisms of stem cell depletion in the airways of transplanted lungs will be a major step forward in understanding OB pathogenesis and will inform the use of stem cells as a therapeutic approach to prevent or delay OB.